1. Field of the Invention
This invention relates to the treatment of patients with neoplastic disorders using compositions containing physiologically stable compounds of butyric acid, butyric acid salts and derivatives, and combinations thereof. These compositions initiate or accelerate the differentiation of neoplastic cells and enhance the surface expression of both MHC and non-MHC antigens on transformed cells promoting their identification and clearance by the immune system. In addition, these same compositions reduce the activity of proteins associated with the development of the multi-drug resistance phenomenon, thereby increasing intracellular concentrations and the effectiveness of conventional chemotherapeutic agents.
2. Description of the Background
The most universally feared disease in the world today is cancer. Cancer has become the laymen's term for all forms of neoplasia including carcinomas, leukemias, tumors and virtually all malignancies. True cancers may better be defined as diseases which have the biological characteristics of malignant neoplasia. A neoplasm is a relatively autonomous growth of tissue, autonomous in that growth does not follow the "rules and regulations" that govern the growth of individual cells of an organism. In other words, growth is in some respect increased. A neoplasm may be benign or malignant. Benign indicates that cell growth is in some way confined, the individual cells are non-invasive and/or highly differentiated, and there is little to no anaplasia. In contrast, malignant neoplasias are non-encapsulated, invasive, and poorly differentiated, grow fairly rapidly, are anaplastic to varying degrees, and metastasize to other areas of the body. In theory, certain benign neoplasias may be early forms of a malignancy or at least a stage along the pathway to malignancy.
Any tissue of a multicellular body that is capable of cell division is capable of becoming cancerous. Cancerous or neoplastic cells act much the same as normal cells. They divide, multiple, process nutrients, perform functions characteristic of their non-neoplastic origins, and they die. Neoplasias become a health concern by carrying out these processes at a higher level effecting the normal functioning of the body. For example, neoplastic cells damage or destroy nearby organs and tissues. Healthy tissue may be out competed for space and/or nutrients by the neoplasm or by neoplastic cells which have metastasized to proximal regions of the body. Neoplasms may also have more systemic consequences effecting the regulation of specific tissues such as those of the immune system.
The basic treatment of neoplastic diseases has remained surprisingly consistent. For confined tumors such as benign hyperplasia, surgery is often suggested and the diseased tissue removed. This approach is generally preferable for otherwise healthy individuals. When the patient is not considered to be a particularly likely candidate for invasive surgical procedures and/or when the neoplasm is unconfined to a single organ or site, for example, has metastasized, drug or radiation therapy is often the only recourse. The principle idea behind these therapies is that all forms of neoplasia involve some degree of increased cell proliferation. Radiation therapy and chemotherapy are successful because they simply and indiscriminately kill multiplying cells. The problem is that not all multiplying cells are neoplastic. Most cells multiply to some degree and the variations in growth rate throughout the body are enormous. Neoplastic cells, although dividing quite rapidly, usually fall somewhere within this range. Consequently, although each of these therapies have been successful in certain contexts, they are usually not a cure.
One popular theory about the biology of cancer is that it represents an arrest in the development of the cell. The cancer cell remains in a relatively immature state and continues to be capable of growth and replication throughout its life. In contrast, a normal cell would mature fully into, for example, a functional bowel cell, blood cell or lung cell, which would not be capable of further proliferation. The normal process is called differentiation and a cancer cell could, therefore, be a cell which has not differentiated fully, quite possibly as a result of oncogene activation. In theory, agents which can force the cancer cell to complete differentiation would render it incapable of further growth and injury to the patient (E. J. Seifert et al., Am. J. Med. 83:757-60, 1987). Agents which can force differentiation of leukemia cells in vitro, such as cytosine arabinoside and retinoic acid, have recently been used effectively in patients to turn leukemia cells into normal, mature-looking cells and slow the course of the disease (L. Sachs, Cancer Res. 47:1981-86, 1987). The phorbal diester 12-0-tetradecanoylphorbol 13-acetate (TPA) has been shown to be an effective inducer of T cell differentiation in acute lymphoblastic leukemia and B cell differentiation in chronic lymphocytic leukemia (J. Cossman et al., N. Engl. J. Med. 307:1251-54, 1982). A naturally occurring tumor promoter, teleocidine B, an indole alkaloid isolated from the mycelia of Streptomyces, has been shown to have similar effects on fibroblasts (A. Bloch, Cancer Treatment Rep. 68:199-205, 1984). Unfortunately, a number of problems including the toxic nature of these inducers, the high dosages which would be required, and the potential for unwanted and dangerous side effects has compromised their usefulness (D. M. Pace et al., Canad. J. Biochem. 45:81-88, 1967).
One differentiation inducer was administered to humans as a therapy against various forms of cancer (J. Watson and M. B. Glasg, The Lancet 618:746-48, Apr. 8, 1933). Crude preparations of butyric acid and butyrate of quanine (kieselguhr and chalk) were used to treat patients suffering from carcinoma of the cervix, rectal cancer, stomach cancer, or papilloma of the ovary. In each case definitive results were undeterminable. Treatment consisted of packing the wounds after a surgical procedure with gelatin capsules containing these substances or simply applying the substance locally. However, in every case, applications were administered in conjunction with surgery and sometimes radiation therapy, both of which themselves would have had a substantial effect on tumors. With combination therapy taken into consideration, the substances had no beneficial effect. In addition, there was no information provided as to doses used or in vivo levels achieved, generally required for any determination of efficacy. Any positive effects observed could be better attributed to the ability of butyric acid to cauterize the afflicted tissue rather than any effect on malignancy. Consequently, it is impossible to determine whether butyric acid played any role and, in fact, the outcome would suggest that it had no positive effect at all.
More recently, preparations of butyric acid were shown to suppress in vitro neoplastic transformation of Syrian hamster cells (J. Leavitt et al., Nature 271:262-65, 1978). These studies demonstrated that aberrant morphology, anchorage-independent growth, and enhanced proteolytic activity, which each correlates with tumorigenicity, were all suppressed after treatments of butyric acid. However, the use of butyric acid as an anti-cancer agent was impractical and untransferable to clinical use. Butyric acid is physiologically unstable. It has an extremely short serum half-life of about two minutes and, more importantly, any biological effect requires a detectable presence. In other words, termination of treatment ends the observed biological effect making its practical application as a pharmaceutical extremely unlikely.
Sodium butyrate, a relatively nontoxic form of butyric acid, but with the same fleeting serum half-life and biological effect, has been shown to force the in vitro differentiation of human erythroleukemia cells, chronic myelogenous leukemia cells, bowel cancer cells, salivary adenocarcinoma cells, pancreatic adenocarcinoma cells, melanoma cells, ovarian adenocarcinoma cells, medullary thyroid carcinoma cells, Burkitt lymphoma cells, astrocytoma cells, and neuroblastoma cells (K. N. Prasad, Life Sci. 27:1351-58, 1980). Differentiation is tied to the expression or repression of a number of different gene products and a number of different biological activities.
For example, sodium butyrate has been shown to cause an increase in the activities of a number of mammalian enzymes in tissue culture including tyrosine hydroxylase, choline acetyltransferase, acetyl cholinesterase, adenylate cyclase in NB cells, adenosine kinase and deaminase, guanosine and adenosine monophosphate kinases, adenine and hypoxanthine phosphoribosyltransferases in human colon carcinoma cells, and sialyl transferase in HeLa cells. Alternatively, other enzymes are inhibited by sodium butyrate. Enzymes whose activities are inhibited include tyrosine transaminase in hepatoma cells, hexokinase and glucokinase in normal liver cells, and lactate dehydrogenase and pyruvate kinase in neuroblastoma cells. Also, other properties of sodium butyrate which have been demonstrated in vitro include the stimulation ganglioside G.sub.M 1 synthesis, induction of expression of .beta.-adrenergic receptors and choleratoxin receptors on HeLa cells, increased production of gonadotropins, and increased synthesis of prostaglandins. Although, the mechanism(s) whereby sodium butyrate forces differentiation of tumors and arrests growth are not fully understood, sodium butyrate does increase intracellular levels of cAMP, inhibit histone acetylation, and inhibit methylation of genomic DNA. In many cases, this differentiation is also accompanied by down-regulation of activated oncogenes in the tumors, but any single specific cause and effect relationship has yet to be established.
Many studies using butyric acid have been performed on HL-60 cells, a human cell line derived from a leukemia and a commonly used myeloid progenitor line. These cells readily differentiate in the presence of about 0.5 mM butyric acid and are used to study the regulation of granulocyte differentiation and cellular metabolism (M. C. Hoessly et al., Cancer Res. 49:3594-97, 1989). Treated cells show phenotypic changes such as the accumulation of numerous granules and condensation of the nucleus (G. Rovera et al., Proc. Natl. Acad. Sci. U.S.A. 76:2779-82, 1979). In addition, transcription of the myb gene is markedly decreased in granulocytic HL-60 cells.
Sodium butyrate, as well as retinoic acid and other retenoids, certain plant lectins, phorbal esters, and cytosine arabinoside, although apparently effective in vitro, were unsuccessful or not developed for in vivo use for a number of reasons. Fairly large amounts of the substances were required to produce a meaningful effect. These levels are difficult to achieve in vivo, or would be toxic, and may produce side effects. Further, although certain agents, such as butyric acid, are fairly well tolerated on their own, the sodium salts were not. Such large amounts of the salt are required that major organ damage attributed to sodium overload was observed in animal studies.
Recent studies have indicated that the action of differentiation inducers may be somewhat definable. Dimethyl sulphoxide (DMSO) or hypoxanthine treatments of Friend leukemia cells were shown to stimulate differentiation and down regulate c-myc expression (E. V. Prochownik and J. Kukowska, Nature 322:848-850, 1986). Using recombinantly produced c-myc to inhibit the reduction of c-myc levels, differentiation of these cells was completely or partially inhibited, indicating that at least partial control of differentiation may reside at the expression of certain oncogenes. Further, as determined by others, butyrate effects the expression of a number of cellular genes. Butyric acid may only indirectly stimulate differentiation by down- or up-regulating the expression of the cellular enzymes which are the direct control. As yet, this is merely speculation and a definitive mechanism of action has still to be determined.
Current modalities for the treatment of malignancy are essentially limited to surgery, radiotherapy or chemotherapy, all of which are relatively nonspecific. Newer approaches aimed directly at the tumor cell are needed. The cellular immune system, with its striking ability to discriminate tumor cells from normal cells, is ideally suited for therapeutic manipulation. There is abundant evidence that the cellular immune system allows immuno-competent animals to reject transplanted tumors or inoculated tumor viruses. Our understanding of the immune mechanisms involved is limited, but directed manipulation of the immune response to tumors by administration of cancer vaccines or lymphokines is feasible. Still, many tumors can readily evade the cellular immune defenses. A major mechanism by which tumor cells escape from surveillance by the cellular immune system in the host is by alteration of Major Histocompatibility Complex (MHC) antigen expression. Down-regulation of MHC expression is extremely common in bowel, neuroblastoma, and small-cell lung tumors. These same tumors commonly express a mutated form of an oncogene which has been shown to down regulate MHC expression. Down-regulation, although a commonly used term, may not be correct. Tumor cells may not be capable of expressing MHC antigens like their mature, normal counterparts because of their immature, undifferentiated state.
The effector arm of the cellular immune system, comprised of cytotoxic T cells (CTL) and natural killer cells (NK), plays a pivotal role in a body's elimination of tumors. The ability to generate a CTL response to a tumor is clearly linked to the ability of an animal to reject that tumor. CTLs directed against tumors are MHC restricted to the target antigen or antigen fragments. Target antigen must be recognized on the tumor cell surface in association with MHC antigens identical, or syngeneic, to those found on the CTL itself HLA and H-2 antigens are the cell surface glycoproteins encoded by the human and murine MHC gene complexes, respectively. This requirement for a syngeneic Class I MHC antigen in association with a tumor or viral antigen is known as dual restriction. MHC expression is also likely to be regulator of tumor recognition by the natural killer arm of the cellular immune system, although its exact role is a subject of much debate. Because of their important role as restriction elements for CT-target cell recognition, the Class I MHC antigens have undergone extensive biochemical analysis. These antigens, referred to as HLA-A, -B, and -C in the human and H-2K, D and L in the mouse, are cell surfaces glycoproteins, each comprised of a polymorphic heavy chain and a non-covalently linked, non-polymorphic light chain, called .beta..sub.2 -microglobulin.
The remarkable dual specificity on the part of the CTL for self (syngeneic MHC) plus tumor antigen, makes it ideally suited to identify and eliminate transformed cells within the animal. Aberrant regulation of MHC expression is, however, a frequent occurrence in human tumors, allowing the tumor circumvent cellular immune surveillance by eliminating one of these two essential recognition elements. Major changes in cellular MHC expression, induced by expression of oncogenes and tumor viruses, have shown that these alterations result in biologically significant resistance to cellular immune cytotoxicity.
The recognition of virus-induced tumor antigens by the cellular immune system has been extensively examined (D. C. Flyer et al., J. Immunol. 135:2287-92, 1985). CTLs directed against sarcoma virus-induced tumor antigens are Class I-restricted. Recognition of the tumor target antigens must be in association with the appropriate syngeneic MHC gene products. Significantly, tumors induced by these viruses control their own immune recognition by direct regulation of the Class I MHC expression of the infected cells (R. T. Maziarz, et al., Mol. Immunol. 27:135-42, 1990). Recent experiments have shown that in many tumor cells, including oncogene-transformed cells, the level of MHC protein expression is so dramatically down-regulated by the infecting tumor virus that antigens cannot be recognized by CTL, which require both syngeneic MHC antigens and viral antigens on the cell surface for recognition, or by CTL lines. The level of CTL-mediated lysis of tumor cells both by MHC-restricted, virus-specific CTL and by CTL directed against allogeneic MHC determinants is directly influenced by the level of MHC Class I antigen expression on the surface of the tumor cells. Induction of the MHC antigens on the surface of the tumor cells made them once again able to be recognized and destroyed by the animal's immune system.
The human erythroleukemia cell line K562, which expresses little or no Class I MHC, is not recognized by human cytotoxic T lymphocytes. By induction of expression of Class I MHC antigens on these tumor cells using interferon, virus, sodium butyrate or transfection, new surface expression of specific Class I MHC antigens in K562 conferred upon these tumor cells susceptibility to both humoral and cellular immune recognition (R. T. Maziarz et al., Cell. Immunol. 130:329-38, 1990). This demonstrates the importance of the repression of the endogenous MHC antigens to the selective survival advantage of such tumors.
In addition to Class I MHC, other antigens are also turned on or their expression increased in the presence of butyric acid. U.S. Pat. Nos. 4,822,821, 4,997,815, 5,025,029, and 5,216,004, all of whose disclosures are specifically incorporated by reference, demonstrate that butyric acid turns on .gamma.-globin synthesis in sickle cell and thalassemic fetal erythrocytes. Other antigens turned on by butyric acid, that are normally present on the surface of mature cells, are often missing from tumor cells. Some of these, like the IL-2 receptor, the EGF receptor or the CEA antigen, are currently being studied for the use as targets for cancer immunotherapy. For example, monoclonal antibodies coupled to diphtheria toxin and directed against the IL-2R or the EGF-R have been developed and are presently being tested. The level of IL-2R expression determines whether the tumors are susceptible to these immune-toxin therapies. Cells not expressing IL-2R are resistant. A vaccine and a toxic antibody against CEA has similarly been developed and is under investigation.
The IL-2 receptor (IL-2R) and its ligand are useful tools for biotherapeutic approaches to a number of hematopoietic malignancies. The IL-2R was first identified as a 55 kd surface peptide, p55, to which the anti-Tac ("anti-T-cell activation" ) monoclonal antibody bound. Later, a 75 kd peptide, p75, and a 64 kd peptide, p64, were identified in the functional IL2R complex and have since been demonstrated to be essential components of the high affinity IL-2R.
The most recent functional analysis of IL2R reveals that the receptor exists in three different isoforms: high affinity for IL-2 (dissociation constant (K.sub.d) of 10.sup.-11 M), intermediate affinity (K.sub.d of 10.sup.-9 M), and low affinity (K.sub.d of 10.sup.-8 M). Low affinity receptors consist of p55 alone and are ineffective in IL2R mediated signal transduction due to failure to internalize the bound ligand. High affinity receptors are heterotrimers (p55, p75, p64) capable of efficient ligand internalization. Intermediate affinity receptors may consist of the heterodimers p55/p75 or p75/p64. The former is capable of binding, but not internalizing ligand, thereby indicating that the p64 component is essential for receptor-mediated internalization of IL2.
Functional high affinity receptors are detected by Scatchard analysis using I.sup.125 -IL-2 on HTLV-1 associated adult T cell leukemia cells and on some chronic lymphocytic leukemia, acute lymphoblastic leukemia, and cutaneous T cell lymphoma (Sezary) cells. Because of the internalization capacity of the high-affinity IL-2R for its ligand, the concept of growth factor receptor-ligand binding as an entry point for the delivery of intracellular toxins has developed. An IL-2R-directed fusion toxin was genetically constructed and consists of a fusion of the membrane-translocating and protein synthesis-inhibiting domains of the diphtheria toxin gene to a full-length IL-2 gene, producing a recombinant protein, DAB486 IL2, capable of selectively targeting and killing high-affinity IL-2R bearing cells. When presented to cells with high affinity IL-2 receptors, the recombinant protein undergoes IL-2R-specific binding and internalization via receptor-mediated endocytosis. Processing occurs within the acidic endosome, and the A fragment of the toxin passes into the cytosol, where it inhibits protein synthesis by ADP-ribosylation of elongation factor-2, ultimately resulting in cell death. Toxin effects can be blocked with excess IL-2, with excess antibodies to IL2R, or with chloroquine which prevents endosomal acidification. A second generation IL-2 fusion toxin, referred to as DAB389-IL2, has been created in which the carboxy-terminal 97 amino acids of DAB486 are deleted, resulting in a shorter protein with a higher affinity for the IL-2R.
Based on toxin killing experiments of established cell lines using both DAB486-IL-2 and DAB389-IL 2, neoplastic cells from patients with high affinity receptors are uniformly intoxicated (minimum inhibitory concentration.sub.50 or MIC.sub.50 of &lt;2.5.times.10.sup.-10 M), while those tumor cells without high-affinity IL2 receptors are unaffected. Previous studies involving treatment of toxin-insensitive, IL-2R negative cells with inducing agents have demonstrated in the case of PHA (phytohemagglutinin; 10 ug/ml) and bryostatin (1.times.10.sup.-7 M), that induction of IL2R, measured by the TAC antibody, is associated with intoxication by the fusion toxins at a MIC.sub.50 similar to that reported for toxin-sensitive ATL cells. Clinical studies using this fusion toxin have been undertaken and clinically significant responses have been reported in 44% of patients with cutaneous T-cell lymphoma, 28% of those with low-grade and intermediate grade non-Hodgkin's lymphoma, and 15% of those with refractory Hodgkin's disease. All responding patients had IL-2 receptor-expressing tumors as measured by CD25 (TAC) staining.
While the p55 (TAC) peptide is expressed on many different hematopoietic malignancies, few other than HTLV-1 associated adult T-cell leukemia cells uniformly express the high-affinity receptor isoform. Since the Il-2R targeted fusion toxin is specifically cytotoxic only for cells expressing the high-affinity IL-2R, its therapeutic potential will be limited to those diseases in which the high-affinity receptor isoform is found. It would be very useful to extend the therapeutic potential of the IL-2 receptor-targeted fusion toxin proteins by developing physiologically inducers of the high-affinity IL-2R to convert non-expressing or low-expressing tumors to the high affinity IL-2R positive, toxin-sensitive state.
The P-glycoprotein (Pgp), encoded by the mdr-1 gene, is the protein responsible for the majority of multi-drug resistance to chemotherapeutic agents in tumors. This molecule appears to function as a pump in the cell membrane, which pumps, among other substances, certain chemotherapeutic agents out of the cell, decreasing their intra-cellular concentrations and limiting their activity.
Treatment of Pgp-expressing multidrug-resistant SW620 cell line with sodium butyrate resulted in active interference with Pgp function. After sodium butyrate treatment in SW620 human colon carcinoma cells, the intracellular accumulation of the chemotherapeutic agents vinblastine, adriamycin, and actinomycin D increased 10-fold. Sodium butyrate, while increasing Pgp levels, inhibited the phosphorylation of Pgp, and blocked the function of this drug-resistance protein.
A condition known as diversion colitis frequently develops in segments of the colorectum after a surgical diversion of the fecal stream. It persists indefinitely unless the excluded segment is reanastomosed. The disease is characterized by bleeding from inflamed chronic mucosa that mimics the bleeding of idiopathic inflammatory bowel disease, and it may culminate in stricture formation. It may represent an inflammatory state resulting from a nutritional deficiency in the lumen of the colonic epithelium and, possibly, may be effectively treated using short-chain fatty acids, the missing nutrients (J. M. Harig et al., N. Engl. J. Med. 320:23-28, 1989).
Intra-rectal butyric acid produces a consistent and reproducible colitis in mice. The severity of response observed was proportional to the concentration of butyric acid utilized. The colitogenic action of butyric acid could not be reproduced by low pH alone, or by the presence of the butyrate anion at neutral or alkaline pH (D. M. McCafferty and I. J. Zeitlin, Int. J. Tissue React. 11:165-68, 1989). However, at lower concentrations, butyric acid may have some beneficial effects. Compositions containing 80 mM acetate, 30 mM propionate, and 40 mM butyrate were used twice daily as rectal irrigations. The compositions induced improvement in nine out of ten patients with distil colitis (R. I. Breuer et al., Int. J. Colorectal Dis. 6:127-32, 1991). In another ten patient study, short-chain fatty-acid irrigations were again found to ameliorate inflammation in diversion colitis in patients unresponsive to other conventional forms of treatment. The histological degree of inflammation decreased, discharge of blood ceased, and endoscopic scores fell. On placebo, all of these parameters remained unchanged (W. Scheppach et al., Gastroenterology 103:1709-10, 1992).